GENETICS OF HERPESVIRUS-HOST CELL INTERACTIONS

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The analysis of the regulatory phenomena controlling the coordinately and sequentially ordered synthesis of herpes simplex virus type 1 (HSV-1) sepcified proteins during infection of cells in culture is the objective of this research. Our aim is to identify the regulatory sequences on viral genes, the regulatory signals to which they respond and the mechanisms by which these interact to control viral gene expression. The viral proteins have been classified into three temporal groups termed Alpha, Beta and Gamma. Alpha proteins act as positive regulatory elements for the expression of Beta and Gamma proteins. The primary experimental tool, in our regulatory studies, is cells transformed for restricted regions of the viral genome. These transformed cells are genetically manipulated by infection with viral mutants for information on expression of the transforming sequences. Cells will be transformed for plasmids carrying an Alpha gene for the regulatory protein ICP4 and a Beta gene for glycoprotein B to look directly at the induction of the Beta gene by the Alpha gene product. This construction will provide a cell capable of expressing only one of the immunogenically active glycoproteins induced in the course of viral infections. Gamma gene regulation will be explored using this system. Cells will be transformed for both the Gamma gC gene and the Beta TK gene for direct comparison of induced expression. The effects of Alpha and Beta gene products and of viral DNA synthesis on the induction of these two classes of genes will be explored. The cells transformed for HSV-1 segments will serve as permissive cells for the isolation of host range mutants. Amber nonsense mutants will also be sought. Purified ICP4 will be examined in vitro for specific binding to regulatory sequences of the gB gene. Sites of binding will be detected by the DNA footprinting technique. The involvement of additional viral and/or cellular factors in specific binding will be explored. The effects of mutant alterations in the ICP4 protein and sequence alteration in the gB regulatory region will be measured. The region spanned by the HSV-1 Eco RI F fragment will be reexamined for morphological and oncogenic transformation capacity. Rat 2 tk- cells have been transformed to tk+ with a plasmid containing the HSV-1 TK gene and the Eco RI F fragment to insure that each transformant has received these viral sequences. The possibility that ICP4 may play a role in morphological transformation will be explored.